Fig6: Effect of leptin injection on islet Trpm5 expression and circulating insulin levels in control-fed and food-restricted mice. Bodyweight, plasma glucose, plasma insulin, and islet Trpm5 expression in ob/ob mice that were injected for 2 days with either 1 mg leptin/kg bodyweight or vehicle. Mice were divided in two groups: they had either free access to food during the course of the experiment (a) or were put on food restriction (b), meaning that they received 3 g of food per mouse per day and were fasted overnight during the last night before islet isolation. Trpm5 expression in islets from leptin-injected mice was normalized to the average Trpm5 expression in islets from vehicle-injected mice that had followed the same food protocol. N = 4 mice per group, **p < 0.01, ***p < 0.001

Mentions:
In order to investigate whether the recovery of the leptin pathway (without altering the metabolic phenotype) would be sufficient to upregulate Trpm5 expression, we injected 9-week-old ob/ob mice for 2 days with either 1 mg leptin per kilogram bodyweight or vehicle. Since leptin will have a dramatic and immediate effect on food intake and consequently on the metabolic phenotype, animals were divided in two groups as follows: one group of mice had free access to food for the whole duration of the experiment (Fig. 6a), whereas a second group was put on food restriction, meaning that they received 3 g of food per day and were fasted overnight during the last night before islet isolation (Fig. 6b). Bodyweight was not altered by this short period of leptin treatment, neither in the fed ad libitum group (vehicle-injected 44.1 ± 1.7 g vs. leptin-injected 42.7 ± 1.1 g; p = 0.52, n = 4 mice per group) nor in the food restriction group (vehicle-injected 41.0 ± 0.7 g vs. leptin-injected 39.2 ± 1.5 g; p = 0.33, n = 4 mice per group). Also, plasma glucose levels did not change after 2 days of leptin treatment for both groups: vehicle-injected 245.5 ± 68.2 mg/dl versus leptin-injected 167.5 ± 4.9 mg/dl; p = 0.30, n = 4 mice per group for the fed ad libitum mice and vehicle-injected 179 ± 18 mg/dl versus leptin-injected 155 ± 17 mg/dl; p = 0.38, n = 4 mice per group for the mice put on food restriction. However, plasma insulin was dramatically decreased by leptin treatment in the fed ad libitum mice (vehicle-injected 16.9 ± 1.4 ng/ml vs. leptin-injected 6.2 ± 1.0 ng/ml; p = 0.0008, n = 4 mice per group). In contrast, the mice that were on food restriction displayed normal insulin levels (4.4 ± 1.6 ng/ml vs. 2.7 ± 1.2 ng/ml, respectively, p = 0.41, n = 4 mice per group). Interestingly, Trpm5 was upregulated by leptin treatment in mice that had free access to food (vehicle-injected 0.96 ± 0.43 vs. leptin-injected 3.65 ± 0.53; p = 0.0092, n = 4 mice per group) but not in mice that were on food restriction (vehicle-injected 0.95 ± 0.36 vs. leptin-injected 1.08 ± 0.31, p = 0.86, n = 4 mice per group). These data strongly suggest that plasma insulin levels are a critical factor in the regulation of islet Trpm5 expression.Fig. 6

Fig6: Effect of leptin injection on islet Trpm5 expression and circulating insulin levels in control-fed and food-restricted mice. Bodyweight, plasma glucose, plasma insulin, and islet Trpm5 expression in ob/ob mice that were injected for 2 days with either 1 mg leptin/kg bodyweight or vehicle. Mice were divided in two groups: they had either free access to food during the course of the experiment (a) or were put on food restriction (b), meaning that they received 3 g of food per mouse per day and were fasted overnight during the last night before islet isolation. Trpm5 expression in islets from leptin-injected mice was normalized to the average Trpm5 expression in islets from vehicle-injected mice that had followed the same food protocol. N = 4 mice per group, **p < 0.01, ***p < 0.001

Mentions:
In order to investigate whether the recovery of the leptin pathway (without altering the metabolic phenotype) would be sufficient to upregulate Trpm5 expression, we injected 9-week-old ob/ob mice for 2 days with either 1 mg leptin per kilogram bodyweight or vehicle. Since leptin will have a dramatic and immediate effect on food intake and consequently on the metabolic phenotype, animals were divided in two groups as follows: one group of mice had free access to food for the whole duration of the experiment (Fig. 6a), whereas a second group was put on food restriction, meaning that they received 3 g of food per day and were fasted overnight during the last night before islet isolation (Fig. 6b). Bodyweight was not altered by this short period of leptin treatment, neither in the fed ad libitum group (vehicle-injected 44.1 ± 1.7 g vs. leptin-injected 42.7 ± 1.1 g; p = 0.52, n = 4 mice per group) nor in the food restriction group (vehicle-injected 41.0 ± 0.7 g vs. leptin-injected 39.2 ± 1.5 g; p = 0.33, n = 4 mice per group). Also, plasma glucose levels did not change after 2 days of leptin treatment for both groups: vehicle-injected 245.5 ± 68.2 mg/dl versus leptin-injected 167.5 ± 4.9 mg/dl; p = 0.30, n = 4 mice per group for the fed ad libitum mice and vehicle-injected 179 ± 18 mg/dl versus leptin-injected 155 ± 17 mg/dl; p = 0.38, n = 4 mice per group for the mice put on food restriction. However, plasma insulin was dramatically decreased by leptin treatment in the fed ad libitum mice (vehicle-injected 16.9 ± 1.4 ng/ml vs. leptin-injected 6.2 ± 1.0 ng/ml; p = 0.0008, n = 4 mice per group). In contrast, the mice that were on food restriction displayed normal insulin levels (4.4 ± 1.6 ng/ml vs. 2.7 ± 1.2 ng/ml, respectively, p = 0.41, n = 4 mice per group). Interestingly, Trpm5 was upregulated by leptin treatment in mice that had free access to food (vehicle-injected 0.96 ± 0.43 vs. leptin-injected 3.65 ± 0.53; p = 0.0092, n = 4 mice per group) but not in mice that were on food restriction (vehicle-injected 0.95 ± 0.36 vs. leptin-injected 1.08 ± 0.31, p = 0.86, n = 4 mice per group). These data strongly suggest that plasma insulin levels are a critical factor in the regulation of islet Trpm5 expression.Fig. 6

Bottom Line:
The glucose-induced Ca(2+) oscillation pattern in db/db and ob/ob islets mimicked those of Trpm5 (-/-) islets.Leptin treatment had no additional effect on Trpm5 expression levels when plasma insulin levels were comparable to those of the vehicle-injected control group.In murine β cell line, MIN6, insulin downregulated TRPM5 expression in a dose-dependent manner, unlike glucose or leptin.